CCL16

HCC-4/CCL16 Joseph A. Hedrick Human Genome Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA corresponding author tel: 908-740-7408, fax: 908-740-7101, e-mail: [email protected] DOI: 10.1006/rwcy.2001.1124. Chapter posted 5 November 2001

SUMMARY

Alternative names

HCC-4 (hemofiltrate CC chemokine-4) (Hedrick et al., 1998), also commonly known as LEC (liver-expressed chemokine), belongs to a group of macrophage inflammatory protein (MIP)-related chemokines and is most similar (38% identity) to HCC-1. HCC-4 is reported to be chemotactic for both monocytes and lymphocytes (Hedrick et al., 1998, Youn et al., 1998), but is less potent than most other chemokines. More recently Howard et al. (2000) reported HCC-4 to be effective in generating cell adhesion, with a potency comparable to RANTES in the assay employed. This suggests that regulation of adhesion may, in fact, be the primary biologic function of HCC-4 (Howard et al., 2000). Cross-desensitization experiments have shown that HCC-4 shares at least one receptor with RANTES (Hedrick et al., 1998) and more recently HCC-4 has been reported to interact with two receptors, CCR1 and CCR8 (Howard et al., 2000). The expression pattern of HCC-4 at the level of mRNA includes the liver, but other sites are controversial, as discussed below.

HCC-4 is also known as LEC (liver-expressed chemokine) (Shoudai et al., 1998), LMC (lymphocyte and monocyte chemoattractant) (Youn et al., 1998), LCC-1 (liver CC chemokine1) (Yang et al., 2000), and NCC-4 (novel CC chemokine4) (Naruse et al., 1996, Shoudai et al., 1998). According to the recently proposed chemokine nomenclature (Zlotnik and Yoshie, 2000), HCC-4 is now designated CCL16.

BACKGROUND

Discovery HCC-4 was originally reported as an uncharacterized, chemokine-like gene present in a chemokine gene cluster on chromosome 17q11.2 (Naruse et al., 1996). Several groups subsequently reported cloning of the cDNA for human HCC-4 (Hedrick et al., 1998, Shoudai et al., 1998, Youn et al., 1998) and in vitro characterization of the protein's biological activity (Hedrick et al., 1998, Youn et al., 1998)

Cytokine Reference

Structure HCC-4 possesses a primary amino acid sequence typical of chemokines. The C terminus is somewhat extended (about 20 amino acids) compared with most chemokines and contains a single consensus site for N-terminal glycosylation. The precise sequence and molecular weight of the mature HCC-4 protein has not been experimentally determined.

Main activities and pathophysiological functions HCC-4 is reportedly chemotactic for a variety of leukocytes including T cells and monocytes, however the concentrations required for maximal effect in in vitro microchemotaxis assays are 10±100 times higher than those typically observed with other chemokines (Hedrick et al., 1998, Youn et al., 1998, Howard et al., 2000). Surprisingly, HCC-4 is more effective in generating cell adhesion than chemotaxis and its effects in this regard were comparable to those of RANTES (Howard et al., 2000). In addition to its regulation of chemotaxis and adhesion, HCC-4 has

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been reported to possess myelosuppressive activity comparable to that of MIP-1 (Youn et al., 1998).

Cells and tissues that express the gene

GENE AND GENE REGULATION

The expression of HCC-4 is somewhat controversial as different groups have reported either liver-specific expression or more ubiquitous expression. Hedrick et al. (1998) reported that a small, approximately 0.5 kb, mRNA was observable in many tissues, while a 1.5 kb mRNA was observed only in the presence of IL-10. Shoudai et al. (1998) also reported both 0.5 kb and 1.5 kb mRNAs, but observed expression only in liver (Shoudai, et al., 1998). Similar liver-specific expression was observed by Yang et al. (2000). The identity of the cells that express HCC-4 in the liver is unknown, however HepG2 hepatoma cells are reported to express HCC-4 in a constitutive manner (Yang et al., 2000).

Accession numbers Human cDNA: NM_004590, U91746, AB007454, XM_008455, AF039955, AF055467, AF039954, AB018249 Human gene: AF088219

Chromosome location The human gene for HCC-4 is present at 17q11.2 (Naruse et al., 1996, Youn et al., 1998; Fukuda et al., 1999). The mouse gene for HCC-4 appears to be a pseudogene (Fukuda et al., 1999).

PROTEIN

Relevant linkages

Sequence

The gene for HCC-4 is separated from the gene for HCC-1 by only 2.2 kb (Fukuda et al., 1999). The HCC-2 gene also resides 12 kb from the HCC-1 gene (Pardigol et al., 1998), thus these three chemokine genes are closely linked.

See Figure 1.

Regulatory sites and corresponding transcription factors The gene for human HCC-4 according to Fukuda et al. (1999) lacks a typical TATA box and transcription initiates at multiple sites (‡30, ‡27, ‡1). Other regulatory sites as noted by Fukuda et al. (1999) are: ÿ320, C/EBP -binding site, ÿ282, Hepatocyte nuclear factor-3 -binding site, ÿ117, C/ EBP -binding site, ÿ45, c/EBP binding site, ÿ44, Hepatocyte nuclear factor-3 -binding site. The 30 flanking region contains two AATAAA sites for transcript termination that are separated by an Alu repetitive sequence. This region also contains a single potential mRNA instability sequence (Hedrick et al., 1998, Shoudai et al., 1998).

Description of protein Mature protein (predicted): 97 amino acids (Hedrick et al., 1998; Shoudai et al., 1998), 100 amino acids (Youn et al., 1998).

Important homologies HCC-4 is a member of the CC chemokine family and is most homologous to HCC-1 (38% identity).

Posttranslational modifications Posttranslational modification of HCC-4 has not been experimentally determined, though a single consensus site for N-linked glycosylation is present in the C terminus (Hedrick et al., 1998; Shoudai et al., 1998; Youn et al., 1998).

Figure 1 Amino acid sequence for HCC-4/CCL16. MKVSEAALSLLVLILIITSA SRSQPKVPEW VNTPSTCCLK YYEKVLPRRL VVGYRKALNC HLPAIIFVTK RNREVCTNPN DDWVQEYIKD PNLPLLPTRN LSTVKIITAK NGQPQLLNSQ

HCC-4/CCL16 3

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Hedrick et al. (1998) have shown that monocytes express message for HCC-4. Low levels of expression were also reported in some T cells and NK cells (Hedrick et al., 1998). HepG2 hepatoma cells are reported to express HCC-4 in a constitutive manner (Yang et al., 2000). To date, expression of HCC-4 has not been studied at the protein level.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The 1.5 kb transcript of HCC-4 has been reported to be stabilized in the presence of IL-10 (Hedrick et al., 1998). Expression of HCC-4 in HepG2 hepatoma cells is upregulated by hypoxic exposure (Yang et al., 2000).

RECEPTOR UTILIZATION HCC-4 has been reported to interact with two receptors, CCR1 and CCR8 (Howard et al., 2000). The affinity of HCC-4 for these receptors has not been determined directly, however Howard et al. (2000) reported IC50 values of 11.8 nM, 24.6 nM, and 49.1 nM for CCR1 based on competition with labeled RANTES, MIP-1, and MIP-1 , respectively. The affinity of HCC-4 for CCR8 is somewhat higher, with an IC50 of 7.1 nM based on competition with labeled I-309 (Howard et al., 2000).

IN VITRO ACTIVITIES

In vitro findings HCC-4 has been shown to regulate chemotaxis of lymphocytes and monocytes in vitro (Hedrick et al., 1998; Youn et al., 1998). HCC-4 also induces calcium flux in human monocytes and THP-1 cells (Hedrick et al., 1998; Youn et al., 1998). Like many other chemokines, HCC-4 also displays myelosuppressive activity (Youn et al., 1998).

Bioassays used Typical assays for chemokine activity have been employed to characterize HCC-4 including

microchemotaxis assays, transwell chemotaxis assays, measurement of intracellular calcium flux, and cellular adhesion.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects As previously mentioned, the normal physiological role of HCC-4 is unclear. At present some disagreement remains regarding where HCC-4 mRNA is expressed and nothing has been published regarding the levels of protein present either in circulation or in specific tissues.

IN THERAPY HCC-4 has not been used in any human studies, however Giovarelli et al. (2000) have recently reported that HCC-4 is effective in a mouse antitumor model. In this model, an aggressive and poorly immunogenic mammary adenocarcinoma line designated TSA-pc was engineered to express human HCC-4. Normally, TSA-pc cells grow quickly in either Balb/c or nu/nu mice, giving rise to lung metastases, and ultimately leading to death with a mean survival time of 21±25 days in the study reported (Giovarelli et al., 2000). Interestingly, Balb/c mice injected with TSA-LEC cells generally failed to develop tumors and those which did had a mean survival time of 67 days. The T deficient nu/nu mice in contrast, still developed tumors though survival time was increased to 34 days. Interestingly, TSALEC cells demonstrated decreased metastatic capacity and were also able to induce protective immunity to subsequent challenge with TSA-pc cells. Antibody blocking experiments in this model suggest that both polymorphonuclear leukocytes and CD8‡ T cells are important in tumor rejection, while NK cells and CD4‡ cells were not significant contributors.

NOTE Nomiyama et al. have recently reported CCL16 interaction with CCR1, CCR2, CCR5 and CCR8. In Nomiyama H, Hieshima K, Nakayama T, Sakaguchi T, Fujisawa R, Tanase S, Nishiura H,

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Matsuno K, Takamori H, Tabira Y, Yamamoto T, Miura R, Yoshie O. (2001). Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes. Int. Immunol. 13, 1021±1029.

References Fukuda, S., Hanano, Y., Iio, M., Miura, R., Yoshie, O., and Nomiyama, H. (1999). Genomic organization of the genes for human and mouse CC chemokine LEC. DNA Cell. Biol. 18, 275±283. Giovarelli, M., Cappello, P., Forni, G., Salcedo, T., Moore, P. A., LeFleur, D. W., Nardelli, B., Carlo, E. D., Lollini, P. L., Ruben, S., Ullrich, S., Garotta, G., and Musiani, P. (2000). Tumor rejection and immune memory elicited by locally released LEC chemokine are associated with an impressive recruitment of APCs, lymphocytes, and granulocytes. J. Immunol. 164, 3200±3206. Hedrick, J. A., Helms, A., Vicari, A., and Zlotnik, A. (1998). Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10. Blood 91, 4242±4247. Howard, O. M., Dong, H. F., Shirakawa, A. K., Oppenheim, J. J. (2000). LEC induces chemotaxis and adhesion by interacting with CCR1 and CCR8. Blood 96, 840±845.

Naruse, K., Ueno, M., Satoh, T., Nomiyama, H., Tei, H., Takeda, M., Ledbetter, D., Van Coillie, E., Opdenakker, G., Gunge, N., Sakaki, Y., Iio, M., and Miura, R. (1996). A YAC contig of the human CC chemokine genes clustered on chromosome 17q11.2. Genomics 34, 236±240. Pardigol, A., Forssmann, U., Zucht, H.-D., Loetscher, P., Schulz-Knappe, P., Baggiolini, M., Forssmann, W.-G., and MaÈgert, H.-J. (1998). HCC-2, a human chemokine: gene structure, expression pattern, and biological activity. Proc. Natl. Acad. Sci. USA 95, 6308±6313. Shoudai, K., Hieshima, K., Fukuda, S., Iio, M., Miura, R., Imai, T., Yoshie, O., and Nomiyama, H. (1998). Isolation of cDNA encoding a novel human CC chemokine NCC-4/LEC. Biochem Biophys. Acta 1396, 273±277. Yang, J. Y., Spanaus, K. S., and Widmer, U. (2000). Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver. Cytokine 12, 101±109. Youn, B. S., Zhang, S., Broxmeyer, H. E., Antol, K., Fraser, M. J. Jr., Hangoc, G., and Kwon, B. S. (1998). Isolation and characterization of LMC, a novel lymphocyte and monocyte chemoattractant human CC chemokine, with myelosuppressive activity. Biochem. Biophys. Res. Commun. 247, 217± 222. Zlotnik, A., and Yoshie, O. (2000). Chemokines: a new classification system and their role in immunity. Immunity 12, 121±127.